Recent advances in molecular biology have greatly accelerated the rate at which genes can be cloned and characterized, rendering determination of the complete genomic sequence of an organism an attainable goal. Accordingly, large scale genomic sequencing efforts for several organisms including bacteria, yeast, and nematodes have already yielded extensive genomic sequence information. Recently, the Human Genome Project, an ambitious project to obtain the entire sequence of the human genome, has commenced. The Human Genome Project paves the way for an even more ambitious project sometimes referred to as the Human Genome Diversity Project, a project aimed at identifying and characterizing allelic differences between humans which manifest themselves in a phenotypically important manner. For example, some allelic variations may be the source of debilitating diseases such as sickle cell anemia or neoplastic diseases.
With cloning techniques well developed, the rate at which information can be extracted from the cloned DNA becomes a limiting factor in determining the genomic sequence of an organism. Accordingly, advances in automated sequencing will reduce the cost and time required to sequence the genomes of model organisms.
For example, in order to sequence the 3 billion nucleotides in the human genome in a ten year span, it is necessary for the automated sequencing devices to achieve a sequencing rate of three hundred million bases per year. At the same time, the sequence information obtained using these automated systems must be accurate, reliable, and efficient without requiring the involvement of highly skilled personnel to a high degree. In addition, the cost of operating and maintaining the automated sequencing devices must be minimized.
In the past, slab gel electrophoresis was used to sequence DNA. (See U.S. Pat. No. 4,811,218 and EPO 0533302A1, the disclosures of which are incorporated herein by reference). However, such techniques are prohibitively limited in the context of genomic sequencing efforts. Recently, capillary electrophoresis has emerged as a viable approach to genomic sequencing. In capillary electrophoresis the detectable products of sequencing reactions conducted on the nucleic acids to be sequenced are applied to small diameter capillary tubes containing a separating medium such as a soluble cellulose derivative or polyacrylamide. A high voltage is applied along the tubes, thereby causing the nucleic acids to migrate along the length of the capillary tubes. As in conventional sequencing techniques, the differential migration rates of nucleic acids of different lengths enables sequence determination. Nucleic acids migrating through the capillary tubes are detected upon reaching a detection region in the capillary tubes using such techniques as laser induced fluorescence.
While capillary electrophoresis permits high resolution of nucleic acids of different lengths and rapid sequence determination, several technical hurdles remain in the application of this technology to genomic sequencing efforts. One important limitation in existing methods is the difficulty in obtaining sequence information from a large number of capillaries simultaneously.
Huang et al. provided a device in which multiple capillaries are arrayed side by side as illustrated in FIG. 1, and are sequentially scanned by a laser and fluorescence is detected using a photomultiplier. (See Huang et al., Anal. Chem. 64:967-972 (1992), the disclosure of which is incorporated herein by reference). However, the effectiveness of the device of Huang is reduced as a result of lightscatter from the capillary walls and the interfaces between the separation medium and the capillaries. Furthermore, in the Huang device, the entire stage on which the capillaries are mounted is linearly translated back and forth underneath the light illumination and collection apparatus, resulting in stress on the capillaries and difficulties in precise position control.
Dovichi et al. provided a device in which multiple rows of capillaries terminate at different levels in a sheath flow cuvette. (See WO 94/29712, the disclosure of which is incorporated herein by reference). Sheath fluid draws individual sample streams through the cuvette. However, the device of Dovichi et al. requires a bubble removing system to ensure that bubbles do not form in the cuvette. To reduce background signal the Dovichi device requires the use of highly purified sheath fluid. In addition, in order to achieve the required sensitivity of signal detection, the Dovichi design requires placement of the laser very close to the termini of the capillaries. Finally, with the Dovichi system it is difficult to adjust the system after each use and to change the capillaries.
For the preceding reasons, there is a need for a detection system which achieves a high throughput while requiring little attention by highly trained personnel.
In one embodiment, the invention comprises an electrophoresis apparatus comprising a plurality of capillaries, wherein each of the plurality of capillaries intersects a plane, and wherein the plurality of capillaries is arranged to intersect the plane such that a curving contour is formed by the intersection points of each of the plurality of capillaries with the plane. Advantageously, such a plurality of capillaries may form a substantially cylindrical array.
Another aspect of the present invention includes an electrophoresis apparatus comprising a plurality of capillaries and a capillary guide. The capillary guide may form a substantially cylindrical shell having an inner surface and an outer surface and may include capillary inputs proximate to a first end and capillary outputs proximate to a second end. The capillaries may then be exposed along a portion of at least one of the inner surface and the outer surface of the capillary guide.
Methods of making electrophoresis apparatus are also provided. In one such embodiment of the invention, a method of making a capillary electrophoresis apparatus comprises the steps of arranging a plurality of capillaries in a substantially cylindrical array and mounting a light collecting lens adjacent to at least one of the plurality of capillaries.
Another aspect of the present invention comprises methods of performing electrophoresis. One such method comprises the steps of rotating an array of capillaries, illuminating capillaries in the rotating array and detecting light emitted by substances in said capillaries. An alternative method of performing electrophoresis comprises the steps of rotating an illuminator past an array of capillaries and detecting light emitted by substances in the capillaries.
Additional electrophoresis apparatus is also provided, including a grid for aligning capillaries with the wells in a microtiter plate. This grid advantageously comprises a body having apertures therein, the apertures being sized for receiving capillaries therein and configured such that the apertures align with the wells in the microtiter plate when the body is placed over the microtiter plate.
Another embodiment of the present invention is an apparatus comprising a first buffer chamber comprising a solid portion, at least one inlet channel in the solid portion, and at least one outlet channel in the solid portion, wherein the at least one inlet channel is in fluid communication with at least one inlet port, the at least one outlet channel is in fluid communication with at least one outlet port and the at least one inlet channel is in fluid communication with the at least one outlet channel. The apparatus also comprises a plurality of capillaries having first ends, second ends, and intermediate portions disposed between the first ends and the second ends, wherein the first ends extend into the first buffer chamber and are in fluid communication with the at least one inlet channel and the at least one outlet channel. In some aspects of this embodiment, the apparatus further comprising a second buffer chamber, wherein the second ends of the capillaries extend into the second buffer chamber. The apparatus may also further comprise an inlet receptacle in fluid communication with the inlet port and an outlet receptacle in fluid communication with the outlet port.
In other aspects of the above embodiment, the apparatus further comprises at least one cap positioned over the first ends of the capillaries, wherein the at least one inlet channel and the at least one outlet channel meet at the at least one cap. The apparatus may have a plurality of inlet channels radiating from a central inlet member and a plurality of outlet channels radiating from a central outlet member.
In other aspects of the above embodiment, the plurality of capillaries is arranged to intersect the plane such that a curving contour is formed by the intersection points of each of the plurality of capillaries with the plane. The curving contour may be substantially closed. The curving contour may form at least a portion of a circle. The capillaries may be arranged to intersect the plane at right angles thereto such that they form a substantially cylindrical array of capillaries.
In some aspects of the above embodiment, the apparatus further comprises an illuminator positioned inside of the substantially cylindrical array of capillaries. The illuminator may be rotatable such that the illuminator sequentially illuminates each capillary in the plurality of capillaries. The apparatus may further comprise a synchronization detector for obtaining data from the capillaries when an optical signal indicating that the capillaries are in focus is detected. The synchronization detector may be a photodetector.
In other aspects of the above embodiment, the apparatus further comprises an illuminator positioned outside of the substantially cylindrical array of capillaries. The substantially cylindrical array of capillaries may be rotatable such that each capillary in the plurality of capillaries is sequentially illuminated by the illuminator. The apparatus may further comprise a synchronization detector for obtaining data from the capillaries when an optical signal indicating that the capillaries are in focus is detected. The synchronization detector may comprises a photodetector.
In another aspect of the above embodiment, the apparatus further comprises a trigger for providing a signal indicative of the beginning of a new scanning cycle.
In another aspect of the above embodiment, the apparatus further comprises a light collector for detecting light emitted by substances within the capillaries, wherein the light collector comprises a spectral separator and at least one single line charge coupled device.
In some versions of the apparatus, the second buffer chamber comprises a grid.
The grid comprises a body having apertures therein, the apertures being sized for receiving capillaries therein and configured such that the apertures align with the wells in a microtiter plate when the body is placed over the microtiter plate. The grid also comprises a reservoir for holding electrophoresis medium. The reservoir for holding electrophoresis medium may comprise a microtiter plate and the grid further comprises a conducting plate positioned between the body and the microtiter plate, the conducting plate having leads thereon which extend into the wells of the microtiter plate.
Another embodiment of the present invention is a method of performing electrophoresis comprising obtaining an electrophoresis device comprising a first buffer chamber comprising a solid portion, at least one inlet channel in the solid portion, and at least one outlet channel in the solid portion, wherein the at least one inlet channel is in fluid communication with at least one inlet port, the at least one outlet channel is in fluid communication with at least one outlet port and the at least one inlet channel is in fluid communication with the at least one outlet channel and a plurality of capillaries having first ends, second ends, and intermediate portions disposed between the first ends and the second ends, wherein the first ends extend into the first buffer chamber and are in fluid communication with the at least one inlet channel and the at least one outlet channel. The method also comprises automatically directing an electrophoresis buffer/separation medium from the at least one inlet port into the first ends of the capillaries such that the capillaries become filled with the electrophoresis/separation medium. In addition, the method also comprises performing electrophoresis on samples introduced into the plurality of capillaries.
In some aspects of the above embodiment, the method further comprises washing the upper buffer chamber by automatically directing a wash solution into the at least one inlet port, from the at least one inlet port into the at least one inlet channel, and from the at least one inlet channel out the at least one outlet port. The method may further comprising opening the at least one inlet port and the at least one outlet port during the washing procedure.
The method may further comprise refilling the capillaries with the electrophoresis/separation medium by automatically directing an electrophoresis buffer/separation medium from the at least one inlet port into the first ends of the capillaries such that the capillaries become filled with the electrophoresis/separation medium. The method may further comprise opening the inlet port during the refilling procedure and closing the outlet port during the refilling procedure.
In some aspects of the above embodiment, the step of performing electrophoresis comprises placing samples into wells in a microtiter plate, positioning a grid having apertures therein over the microtiter plate such that the capillaries extend through the apertures and into the wells of the microtiter plate, and applying a voltage between the first buffer chamber and the second buffer chamber such that the samples move from the wells of the microtiter plate into the capillaries. The grid may further comprise a conducting plate having leads extending therefrom between the grid and the microtiter plate such that the leads extend into the microtiter plate. The method may further comprising filling the wells of the microtiter plate with electrophoresis buffer after the samples have entered the capillaries.